Method for Performing a Blood Count and Determining the Morphology of a Blood Smear

ABSTRACT

A method for counting blood cells in a sample of whole blood. The method comprises the steps of:
         (a) providing a sample of whole blood;   (b) depositing the sample of whole blood onto a slide, e.g., a microscope slide;   (c) employing a spreader to create a blood smear;   (d) allowing the blood smear to dry on the slide;   (e) measuring absorption or reflectance of light attributable to the hemoglobin in the red blood cells in the blood smear on the slide;   (f) recording a magnified two-dimensional digital image of the area of analysis identified by the measurement in step (e) as being of suitable thickness for analysis; and   (g) collecting, analyzing, and storing data from the magnified two-dimensional digital image.       

     Optionally, steps of fixing and staining of blood cells on the slide can be employed in the method.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for performing a blood count by meansof a blood smear.

2. Discussion of the Art

Automated counting of blood cells typically involves counting bloodcells after a sample of whole blood having a known volume is obtainedand subsequently diluted in an appropriate diluent. Knowledge of theinitial volume of the sample and the degree of subsequent dilutionallows a quantitative determination of the numbers of different types ofcells in the given volume of the original sample of whole blood. Forexample, if a microliter of whole blood is diluted so as to yield avolume of 1000 microliters, the dilution ratio is said to be 1:1000, andthe dilution factor is said to be 1000. If a blood count for thisdiluted sample of blood indicates that there are 5000 red blood cellsper microliter, the red blood cell count in the original undiluted bloodsample is equal to the product of 1000 and 5000, i.e., 5,000,000. Thus,the actual blood count of the undiluted sample is 5,000,000 red bloodcells per microliter.

Several physical methods for detecting and enumerating blood cells havebeen employed, such as, for example, analysis of the impedancecharacteristics of the blood cells by means of either direct current orradio frequency signals, the use of optical flow cytometry, whereincells, which are either stained or in their near native state, areexamined by means of light scatter characteristics, absorbancecharacteristics, fluorescence characteristics, or any combination of theforegoing. It has also been suggested that blood cells can be quantifiedby means of direct imaging of the blood cells in combination withanalysis of microscopic images of the blood cells via flow cytometry orwhile the blood cells are suspended in a chamber having specifieddimensions. Instruments have been developed in which either diluted orundiluted samples of blood can be introduced into a counting chamber,the dimensions of which are known, and a blood count can be generated byanalysis of digital images. All of these approaches can be used togenerate the parameters of a blood count.

After a blood count has been completed by one of the aforementionedmethods, a number of the blood samples typically require additionalanalysis by means of a process that involves preparation, staining, andexamination of a blood smear. The process of analyzing a blood smear canemploy a variety of techniques, including manual, automated, orsemi-automated techniques. The analysis of a blood smear can be used toconfirm the accuracy of a blood count, to detect potential interferingsubstances, and to detect some of the fine sub-cellular features ofcells that cannot be detected or interpreted by conventional analyses ofa blood count.

Blood cells are not homogeneous. Blood cells contain sub-cellularfeatures that are smaller than the cells themselves. Such sub-cellularfeatures include nuclei, nucleoli, granules, and cell membranes.Particular examples of analyses of sub-cellular features includeexamination of the shapes of the red blood cells and variations in theshapes of the red blood cells. For example, it is possible to determinethe ratio of the size of the nucleus of the cell to the size of the cellitself by measuring the cross sectional area of each (i.e., the nucleusof the cell and the cell itself) and dividing the measured values. Thisratio, and various other parameters, can be used to determine the degreeof normality of a blood cell.

Potential interfering substances include, but are not limited to, sicklecells, lyse-resistant red blood cells, cells that aggregate for variousreasons, nucleated red blood cells, and unusually high lipidconcentrations. Generally, these interfering substances areabnormalities in the structure(s) of blood constituent(s), whichabnormalities alter the normal reflective and absorptive characteristicsof blood constituents, which normal characteristics enable themeasurements of blood parameters.

With respect to analysis of a blood smear, after a blood smear isprepared, the blood smear can be stained by means of at least oneappropriate stain to identify the morphological characteristics of theblood cells and sub-cellular features of the blood cells. The process ofidentification can be manual or automated. Typically, a stained bloodsmear is examined by a human morphologist, who subjectively assesses themorphological appearances of the cells to provide either quantitativeimpressions of the proportions of different leukocytes orsemi-quantitative impressions of the degree of morphologicalabnormality. Attempts have also been made to automate the process ofanalyzing a blood smear by means of automated microscopes and softwareto recognize patterns in digital images to not only classify leukocytesbut to also provide an interpretation of the morphological changes.

Thus, the performance of a blood count and the subsequent morphologicalanalysis of a blood sample require discrete steps that may involveprocessing the sample of blood through an automated blood countingdevice, forming and staining of a blood smear of the blood sample,either manually or by means of an automated device, followed bymorphological review of the stained blood smear, either manually or bymeans of an automated device.

Although the practices previously described are in widespread use, andalthough the semi-quantitative assessment of cells is possible by amorphological review, performing a quantitative complete blood count ona blood smear has never been suggested. Such a process has two inherentlimitations. When a sample of blood is spread to form a blood smear, thevolume of blood used to form the blood smear cannot be sufficientlycontrolled to a point where an accurate estimate of the volume of bloodcan be made, with the result that the absolute number of cells presentin the blood smear cannot be determined. Furthermore, although devicesin which a monolayer of a blood sample can be deposited have beendeveloped, these devices typically rely on centrifugation to distributecells evenly across the surface of a rectangular-shaped microscopeslide. In FIG. 1A, a microscope slide is designated by the referencenumeral 10, and a drop of blood is designated by the reference numeral12 a. In FIG. 1 B, the microscope slide is designated by the referencenumeral 10, and the blood smear is designated by the reference numeral12 b. The arrow 14 represents the direction of rotation of themicroscope slide 10 during the centrifugation process. Typically, someunknown volume of the blood sample is lost from the microscope slideduring the centrifugation process. Because the quantity of cells lost isunknown and unpredictable, an accurate estimate of the volume of bloodremaining on the microscope slide at the end of the analysis cannot bemade. Therefore, only limited information can be derived with respect tothe proportions of cells in the blood sample, and no information thatrequires knowledge of the total volume of the blood sample can be made.In effect, no measurements for determining the concentration of cellscan be made.

There are two alternative approaches currently used for preparing bloodsmears. The first approach, which is not in widespread use, is the coverslip method. In this method, a drop of a blood sample is placed on amicroscope slide. This drop is covered with a cover slip, and the bloodsmear is subsequently formed by moving the microscope slide and coverslip in opposite directions, thereby effectively smearing the sample. InFIG. 2A, a microscope slide is designated by the reference numeral 20, adrop of blood is designated by the reference numeral 22 a, and a coverslip is designated by the reference numeral 24. In FIG. 2B, themicroscope slide is designated by the reference numeral 20, the bloodsmear is designated by the reference numeral 22 b, and the cover slip isdesignated by the reference numeral 24. The arrow 26 represents thedirection of movement of the cover slip 24.

The second approach, which is much more widely used, is the wedge orpush smear. In this method, a drop of a blood sample is placed on afirst glass slide, typically a microscope slide. A second glass slide,which is termed a smearer or spreader, is first placed downstream of thedrop of the blood sample and is then drawn back to the drop of the bloodsample, whereby the drop of the blood sample is spread across the lineof contact between the drop of the blood sample and the second glassslide. The second glass slide, i.e., the spreader, is then propelledforward, i.e., in the downstream direction, in a single rapid, butgentle, linear motion, whereby the drop of the blood sample is draggedbehind the spreader, thereby forming a blood smear. See, for example,Automatic Working Area Classification in Peripheral Blood Smears UsingSpatial Distribution features Across Scales, W. Xiong, et al.; LET'SOBSERVE THE BLOOD CELLS, D. Tagliasacchi, et al., April 1997,http://www.funsci.com/fun3_en/blood/blood.htm, incorporated herein byreference. In FIG. 3A, a first glass slide is designated by thereference numeral 30, a drop of blood is designated by the referencenumeral 32 a, and the second glass slide, i.e., the spreader, isdesignated by the reference numeral 34. In FIG. 3B, the first glassslide is designated by the reference numeral 30, and the blood smear isdesignated by the reference numeral 32 b. The arrow 36 represents thedirection of movement of the second glass slide 34. In the resultingblood smear, the blood sample is deposited on the first glass slide in awedge in which the thick end of the wedge is positioned at the point ofinitial contact of the drop of the blood sample on the first glassslide, and the thin end of the wedge, which is positioned downstream ofthe thick end of the wedge, contains a monolayer of cells. However, thewedge or push smear requires that the morphological analysis be confinedto the area of the blood smear in which the cells are distributed verythinly in a true monolayer or in a near monolayer. In FIG. 4, amicroscope slide is designated by the reference numeral 40. The thickportion of the blood smear is designated by the reference numeral 42,the thin portion of the blood smear is designated by the referencenumeral 44, and the part of the blood smear suitable for counting cells,i.e., the true monolayer or near monolayer, is designated by thereference numeral 46. In the thick portion of the blood smear, the cellsmay overlay one another to such an extent that an automated instrumentor a human morphologist is unable to reliably identify and record themorphology of the cells. Cells distributed in the upper layers tend toocclude the two-dimensional images of the cells in the lower layers,when the cells are viewed from above. To an observer, when the edges ofcells overlap, the multiple layers of cells appear as a single large,irregularly shaped area. For example, two-dimensional imaging algorithmshave difficulty in discerning the difference between two smalloverlapping cells and one larger cell having an irregular shape. Thisproblem appears to negate the ability to perform a quantitative analysisof the numbers of leukocytes, erythrocytes, and platelets in a bloodsmear, because the area of a blood smear that is suitable for cellcounting would vary unpredictably from blood sample to blood sample withrespect to the thickness and length of the blood smear. Such variationsare shown in FIGS. 5A, 5B, 5C, and 5D. In FIG. 5A, the slide isdesignated by the reference numeral 50 a, and the blood smear isdesignated by the reference numeral 52 a ; in FIG. 5B, wherein the bloodsmear exhibits a difference in shape from the blood smear shown in FIG.5A, the slide is designated by the reference numeral 50 b, and the bloodsmear is designated by the reference numeral 52 b ; in FIG. 5C, whereinthe blood smear exhibits a difference in length from the blood smearshown in FIG. 5A, the slide is designated by the reference numeral 50 c,and the blood smear is designated by the reference numeral 52 c ; inFIG. 5D, wherein the blood smear exhibits a difference in breadth fromthe blood smear shown in FIG. 5A, the slide is designated by thereference numeral 50 d, and the blood smear is designated by thereference numeral 52 d. In summary, even if the same volumes of bloodsamples were used to form blood smears, the areas being evaluated forcounting cells would differ from sample to sample. The principal factorfor determining the thickness and length of a blood smear would likelybe the overall viscosity of the sample, which, in turn, is likely to bedetermined primarily by the concentration of hemoglobin in the sample.

Additional information relating to methods for examining blood smearscan be found at, for example, Peripheral Blood smear—ClinicalMethods—NCBI Bookshelf, Clinical methods, The History, Physical, andLaboratory Examinations, Third edition, H. Kenneth Walker, W. Dallashall, J. Willis Hurst, Butterworths, Peripheral Blood Smear, Edward C.Lynch, http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cm&part=A4584;Hematology Laboratory: Proper Preparation of a Peripheral Blood Smear,Slide Staining with Wright's Stain; Now peripheral blood smearspreparation doesn't depend on laboratory technician's mastery,Scientific and practical magazine <<Clinical laboratoryconsultation>>No. 6, February, 2005: yahoo answers,http://ansers.yahoo.com/question/index?qid=20090824032133AAEtfnf; andEvaluation of the Blood Smear, M. Christopher, University of CaliforniaDavis, Department of Pathology, Microbiology and Immunology School ofVeterinary Medicine, Davis Calif., USA,http://www.vin.com/proceedings/Proceedings.plx?CID=WSAVA2004&PID=8610&Print=1&O=Generic,all of which are incorporated herein by reference.

SUMMARY OF THE INVENTION

In one aspect, this invention provides a method for counting blood cellsin a sample of whole blood. The method comprises the steps of:

-   -   (a) providing a sample of whole blood;    -   (b) depositing the sample of whole blood onto a slide, e.g., a        microscope slide;    -   (c) employing a spreader to create a blood smear;    -   (d) allowing the blood smear to dry on the slide;    -   (e) measuring absorption or reflectance of light attributable to        the hemoglobin in the red blood cells in the blood smear on the        slide;    -   (f) recording a magnified two-dimensional digital image of the        area of analysis identified by the measurement in step (e) as        being of suitable thickness for analysis; and    -   (g) collecting, analyzing, and storing data from the magnified        two-dimensional digital image.        Optionally, process steps for fixing and staining of blood cells        on the slide can be used in the aforementioned method.

It is preferred that the sample of whole blood be a sample of mixedwhole blood. The volume of the sample of whole blood used to form theblood smear can be determined either directly by applying a known volumeof the blood sample to the slide, or indirectly by determining thevolume of the blood applied to the slide by means of a measurement ofoptical density or reflectance of the blood smear on the slide andconverting the value so obtained to a volumetric measure by means of anindependent measurement of hemoglobin made by an instrument, such as,for example, an automated hematology analyzer or a spectrometer.

The concentration of hemoglobin in a sample of whole blood can bedetermined directly at the point of aspiration, typically by means ofreflectance measurement of the blood sample in an optically clearsampling probe, such as, for example, a glass capillary tube.Alternatively, the concentration of hemoglobin in a sample of wholeblood can be determined directly by means conventional absorbancemeasurements following dilution of the sample. In still anotheralternative, the concentration of hemoglobin in a sample of whole bloodcan be determined by measuring absorbance or reflectance of lightattributable to hemoglobin in the red blood cells in a blood smear on aslide. When the concentration of hemoglobin is known, the blood smearcan be scanned by a low power imaging device to determine the opticaldensity of the blood in the blood smear. Because this measurement wouldeffectively determine the amount of hemoglobin in the sample of wholeblood used to form the blood smear, the volume of whole blood that wasactually aspirated and deposited on the slide can be calculated.

In another aspect, this invention provides a device for counting bloodcells in a sample of whole blood. The device comprises:

-   -   (a) a holder for presenting a container containing a sample of        whole blood to an aspiration/dispensing device, an        aspiration/dispensing device for withdrawing a sample of whole        blood from the container and depositing the sample of whole        blood onto a slide, e.g., a microscope slide;    -   (b) a spreader for spreading the sample of whole blood across        the slide to create a blood smear;    -   (c) a dryer for drying the blood smear on the slide;    -   (d) a first imaging system capable of measuring the absorption        or reflectance of light on account of the hemoglobin in the red        blood cells in the blood smear on the slide;    -   (e) a second imaging system capable of recording a magnified,        two-dimensional digital image of the area of analysis identified        by the first imaging system as being of suitable thickness for        analysis; and    -   (f) a computer to collect, analyze, and store results of the        magnified two-dimensional digital image.        Optionally, the device can employ a positioner for positioning        the slide to enable further processing of the blood smear.

The method and the device described herein can consolidate the processof blood counting and review of a blood smear in a single instrument.The method and device described herein require only a few reagents,which reagents are inexpensive. The method and device described hereinare not complex in a technological sense, because only a singleundiluted volume of whole blood is used.

The method and device described herein can detect abnormalities that arecurrently undetectable by conventional hematology analyzers. Suchabnormalities include abnormal red blood cell associations (Rouleaux andaggregation), red blood cell inclusion bodies such as Howell-Jollybodies and malarial parasites. The method and device described hereincan also show sub-cellular changes in the white blood cells, such as theAuer rods seen in acute myeloid leukemias or nucleoli seen in blastcells. Finally, the method and device described herein can detect plasmaabnormalities, such as, for example, increases in protein levels, whichcan be seen in cases of paraproteinemia.

The analysis of the blood count and the blood smear can be performed onthe same sample of whole blood, thereby giving the user the opportunityto directly review the instrument's interpretation of the classificationof cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate one method of preparing a blood smear, i.e.,a centrifugal method. FIG. 1A is a top plan view of a slide showing adrop of whole blood deposited on the slide prior to centrifugation. FIG.1B is a top plan view of the slide of FIG. 1A showing a blood smearformed by means of the centrifugation method.

FIGS. 2A and 2B illustrate a second method of preparing a blood smear,i.e., a cover slip method. FIG. 2A is a top plan view of a slide showinga drop of whole blood deposited on the slide prior to formation of ablood smear. FIG. 2B is a top plan view of the slide of FIG. 2A showinga blood smear formed by means of the cover slip method.

FIGS. 3A and 3B illustrate a third method of preparing a blood smear,i.e., a wedge or push method. FIG. 3A is a side view in elevation of aslide showing a drop of whole blood deposited on the slide prior toformation of a blood smear. FIG. 3B is a top plan view of the slide ofFIG. 3A showing a blood smear formed by means of the wedge or pushmethod.

FIG. 4 is a top plan view of a slide illustrating a blood smearexhibiting a wedge, wherein the wedge has three different regions, inwhich the blood smear in one region is thicker than desired, the bloodsmear in a second region is thinner than desired, and the blood smear ina third region is actually desired for morphological review.

FIGS. 5A, 5B, 5C, and 5D are top plan views of blood smears on slidesillustrating how blood smears vary with respect to their length,breadth, and shape.

FIGS. 6A, 6B, and 6C are top plan views of a blood smear on a slideillustrating how a low power scan of the optical density of the entirearea of a blood smear can be used, in combination with the known volumeof the sample of whole blood used to form the blood smear, to calculatethe volume of the portion of the sample of whole blood contained withinthe portion of the blood smear used to carry out the method describedherein.

FIG. 7 is a schematic diagram illustrating the dispensing of a knownvolume of a sample of whole blood onto a slide.

FIGS. 8A, 8B, 8C, and 8D illustrate how blood smears having differentthickness profiles and concentrations of hemoglobin can be scanned bymeans of low power imaging to establish the volumetric distribution ofthe blood sample across the blood smear, thereby enabling determinationof the volume of blood contained within the area of the blood smearexamined for morphology and cell counting. FIGS. 8A and 8B are top planviews of ten (10) slides, each slide having a blood smear formedthereon. FIG. 8C consists of ten (10) graphs, one graph for each slide,illustrating profiles of the blood smears shown in FIGS. 8A and 8B. FIG.8D is a graph illustrating a profile of a blood smear. The graph in FIG.8D designates the portion of the blood smear that is eligible forreview, i.e., the portion in which blood cells are counted. FIG. 8E is agraph illustrating how the determination of optical density by means ofscanning is shown to correlate with estimates of hemoglobinconcentration made by means of a conventional automated hematologyanalyzer.

DETAILED DESCRIPTION

As used herein, the expression “whole blood” means a living tissue thatcirculates through the heart arteries, veins, and capillaries carryingnourishment, electrolytes, hormones, vitamins, antibodies, heat, andoxygen to the body's tissues. Whole blood contains red blood cells,white blood cells, and platelets suspended in a fluid called plasma. Asused herein, the expression “sample of blood” is synonymous with theexpression “blood sample.” As used herein, the expression “sample ofwhole blood” is synonymous with the expression “whole blood sample.” Asused herein, the expression “blood smear” means a thin film of bloodprepared for the purpose of microscopic image analysis of the individualcells contained therein, usually on a microscope slide, and optionallystained or mixed to impart permanency. As used herein, the expression“sample of mixed whole blood” means a sample of whole blood that hasbeen mixed to resuspend cells in a homogeneous mixture. Blood cells fromsample of whole blood drawn directly from a patient, in the absence offurther processing, tend to settle over a period of time after beingdrawn. Accordingly, the sample of whole blood is mixed prior to beingtested. As used herein, the expression “complete blood count” means atest requested by a doctor or other medical professional that givesinformation about the cells in a patient's blood. The cells thatcirculate in the bloodstream are generally divided into three types:white blood cells (leukocytes), red blood cells (erythrocytes), andplatelets (thrombocytes). For additional information, see Complete bloodcount—Wikipedia, the free encyclopedia,http://en.wikipedia.org/wiki/Complete_blood_count, incorporated hereinby reference. As used herein, the term “slide” means a small glass platefor mounting specimens to be examined under a microscope.

As used herein the term “metameter” means a transformed value, incontrast to one that is directly measured. As used herein, theexpression “red cell distribution width” is a measure of the health ofthe red blood cell population according to the distribution of cellsizes. If the cell sizes of a population of red blood cells from a givensample of whole blood are measured and plotted in a histogram with thenumber of cells of a given size as a function of the size of the cells,the result is the red cell distribution of sizes of the cell, which isapproximately a normal distribution. Accordingly, the expression “redcell distribution width” means the quotient of the standard deviation ofthe distribution of red blood cells divided by the mean of thedistribution of red blood cells, usually multiplied by 100% to convertthe quotient to a percent (%).

As used herein, the expression “mean corpuscular volume” (MCV) means theaverage volume of a red blood cell, measured in femtoliters. As usedherein, the expression “mean corpuscular hemoglobin concentration”(MCHC) means the average amount of hemoglobin in a given volume of redblood cells, measured in percent (%). As used herein, the expression“concentration of hemoglobin” means the amount of hemoglobin in a volumeof blood, measured in g/dL. As used herein, the expression “meancorpuscular hemoglobin” (MCH) means the average amount of hemoglobin inthe average red blood cell, measured in picograms.

As used herein, the expression “cytoplasmic ratio” means the ratio ofthe volume of the cytoplasm in a cell to the total volume of the cell.As used herein, the expression “nuclear ratio” means the ratio of thevolume of the nucleus in a cell to the total volume of the cell.

As used herein, the expression “line of contact” refers to the processof forming a wedge or push smear. In order to form this type of bloodsmear, a drop of a sample of whole blood is placed near an end of aglass slide. A smearer or spreader having a straight edge is touched tothe glass slide and pushed into the drop of whole blood, therebyspreading the drop across the line of contact between the smearer or thespreader and the glass slide. Then the direction of the smearer or thespreader is reversed, and the smearer or spreader is pulled along thelength of the glass slide, thereby pulling a line of the drop of wholeblood with it. The blood is spread on the glass slide in a film until itis depleted, with the result that a wedge of blood remains on the slide.As used herein, the term “wedge” refers to the observation that the filmof blood, i.e., the blood smear, is thicker at one end of the slide thanit is at the other. Viewed from above, the film of blood, i.e., theblood smear, appears to be a rectangle. However, the rectangle is darkerat one end of the glass slide and its color becomes progressivelylighter towards the other end of the glass slide as the film of blood,i.e., the blood smear, becomes thinner.

As used herein, the term “absorbance” means optical density. Absorbanceis represented by the formula

A _(λ)=−log₁₀(I/I ₀)

where I represents the intensity of light at a specified wavelength λthat has passed through a sample (transmitted light intensity) and I₀represents the intensity of the light before it enters the sample orincident light intensity. Absorbance measurements are often carried outin analytical chemistry, because the absorbance of a sample isproportional to the thickness of the sample and the concentration of theabsorbing species in the sample, in contrast to the transmittance I/I₀of a sample, which varies logarithmically with thickness andconcentration.

As used herein, the term “reflectance” means a measure of the incidentelectromagnetic radiation that is reflected by a given interface. It isclosely related to reflectivity but reflectance is more applicable tothin reflecting objects. Reflectance can vary for thin objects due tovariations in the surface thickness and approaches the reflectivity asthe surface becomes thicker. The reflectance may be calculated bycomparing the amount of reflected radiation to the amount of incidentradiation.

As used herein, the expression “low power imaging” and the like refersto imaging wherein a 10× objective lens of a microscope is employed. Asused herein, the expression, “high power imaging” and the like refers toimaging wherein a magnification of 40× to 100× is employed. It should benoted that a magnification of 40× does not require oil immersion, whilea magnification of 100× preferably employs oil immersion. Oil immersionis a technique used to increase the resolution of a microscope.Increased resolution is achieved by immersing both the objective lensand the specimen in a transparent oil of high refractive index, therebyincreasing the numerical aperture of the objective lens. Oil immersionis described in greater detail in Oil immersion—Wikipedia, the freeencyclopedia, http://en.wikipedia.org/wiki/Oil_immersion_objective,incorporated herein by reference. It should be noted that when low powerimaging is used in the method and apparatus described herein, highresolution is not required, because the purpose of low power imaging isto determine the portion of the slide where cells are to be counted. Theportion of the slide where cells are to be counted is a true monolayeror a nearly true monolayer. In contrast, it should be noted that whenhigh power imaging is used in the method and apparatus described herein,a higher resolution than that provided by the low power imaging featuresis required so that the various types of blood cells can be countedindividually. It should also be noted that “low power imaging” issynonymous with relatively low magnification (e.g., 10×) and that “highpower imaging” is synonymous with relatively high magnification (e.g.,40× to 100×). Magnifications other than those set forth herein can beused, so long as the high power imaging provides a substantially highermagnification than does the low power imaging, i.e., at least about 4:1,along with adequate resolution. Additional discussion of magnificationand other characteristics of microscopes, such as, for example,resolution, can be found in Microscope-Wikipedia, the free encyclopedia,http://en.wikipedia.org/wiki/Microscope, incorporated herein byreference, and Microscopy—Wikipedia, the free encyclopedia,http://en.wikipedia.org/wiki/Microscopy, incorporated herein byreference.

The method described herein comprises the steps of:

-   -   (a) providing a sample of whole blood;    -   (b) depositing the sample of whole blood onto a slide, e.g., a        microscope slide;    -   (c) employing a spreader to create a blood smear;    -   (d) allowing the blood smear to dry on the slide;    -   (e) measuring absorption or reflectance of light attributable to        the hemoglobin in the red blood cells in the blood smear on the        slide;    -   (f) recording a magnified two-dimensional digital image of the        area of analysis identified by the measurement in step (e) as        being of suitable thickness for analysis; and    -   (g) collecting, analyzing, and storing data from the magnified        two-dimensional digital image.        Optionally, process steps for fixing and staining of blood cells        on the slide can be used in the aforementioned method. Based on        the aforementioned method, it follows that a device for carrying        out the method comprises:    -   (a) a holder for presenting a container containing a sample of        whole blood to an aspiration/dispensing device, an        aspiration/dispensing device for withdrawing a sample of whole        blood from the container and depositing the sample of whole        blood onto a slide, e.g., a microscope slide;    -   (b) a spreader for spreading the sample of whole blood across        the slide to create a blood smear;    -   (c) a dryer for drying the blood smear on the slide;    -   (d) a first imaging system capable of measuring the absorption        or reflectance of light on account of the hemoglobin in the red        blood cells in the blood smear on the slide;    -   (e) a second imaging system capable of recording a magnified,        two-dimensional digital image of the area of analysis identified        by the first imaging system as being of suitable thickness for        analysis; and    -   (f) a computer to collect, analyze, and store results of the        magnified two-dimensional digital image.        Optionally, the device can include a positioner for positioning        the slide to enable further processing of the blood smear.

The major dimension of the magnified two-dimensional image is typicallyparallel to the longer edge of the slide and the minor dimension of themagnified two-dimensional image is typically parallel to the shorteredge of the slide.

The blood cells in the blood smear can be stained with, for example,cytochemical stains, such as, for example, Wright's stain,May-Grunwald-Giemsa stain, new methylene blue, Field's stain, peroxidaseor fluorescent staining, before or after spreading. In some instances,staining procedures are not required.

If staining is required, the slide can be processed appropriately beforebeing delivered to the imaging component(s) of the device. The imagingcomponent(s) scans the entire slide, or a selected area(s) of the slide,at an appropriate level of magnification for the resolution required toperform the analysis. Imaging component(s) suitable for use herein is(are) described inhttp://www.aperio/com/pathology-services/index-solutions-software.asp,incorporated herein by reference.

If a spreader by which the cells can be distributed in a monolayer isused, the total area covered by the sample of blood in the blood smearcan be determined. The cellular elements in the entire slide can then becounted to provide a complete blood count. Alternatively, the cellularelements in only a portion of the area covered by the monolayer of thesample can be counted, and appropriate calculations can be carried outto determine a complete blood count based on the portion of the entireblood smear that is scanned. FIGS. 1A and 1B illustrate a conventionalmethod for distributing cells in a monolayer.

If a wedge or push approach for creating a blood smear is used, a scanof the optical density or light absorbance of the blood smear can beperformed. The value of the optical density or absorbance for a givenarea of the blood smear is proportional to the thickness of the bloodsmear along the length and width of the blood smear for that given area.Knowledge of the total optical density of the blood smear can then beused to calculate the volume of the blood contained within the area ofthe blood smear that can be reviewed reliably. This process addressesthe differences between blood smears resulting from differences inoverall dimensions of different blood smears, and the areas suitable formorphological review, by recording the value of the optical densitymeasured in a particular portion of the blood smear (e.g., a portion ofthe total area of the blood smear), comparing that value to the opticaldensity of the entire blood smear (i.e., the total area of the bloodsmear), and then determining the blood count on the basis of the ratioof the value of the optical density measured in the particular portionof the blood smear (e.g., a portion of the total area of the bloodsmear) to the optical density of the entire blood smear (i.e., the totalarea of the blood smear) while accounting for the volume of bloodforming the blood smear (i.e., a value that is known or a value that canbe determined from the total area of the blood smear). In a given bloodsmear, the portion of the blood smear that is thick has a higherconcentration of hemoglobin than does the portion of the blood smearthat is thin. Moreover, the portion of the blood smear that is thick hasa higher volume of blood than does the portion of the blood smear thatis thin. It would be expected that more white blood cells would be seenper unit area in the thick portion of the blood smear than would be seenper unit area in the thin portion of the blood smear. In other words, ifa portion of a given blood smear (i.e., the thick portion) is twice asthick as another portion of the given blood smear (i.e., the thinportion), it would be expected that twice as many white blood cellswould be seen in the thick portion of the blood smear as would be seenin the thin portion of the blood smear.

Referring now to FIGS. 6A, 6B, and 6C, which illustrate an example of ablood smear, a slide is designated by the reference numeral 60, thethick portion of the blood smear is designated by the reference numeral62, the thin portion of the blood smear is designated by the referencenumeral 64, and the usable portion of the blood smear is designated bythe reference numeral 66. The entire blood smear is divided into smallersections 68 by means of, for example, a plurality of grid lines parallelto the X-axis and a plurality of grid lines parallel to the Y-axis. Ascan of the slide 60 indicates the optical density of each smallersection 68 of the slide 60. A number representing the optical density ofeach smaller section 68 is imprinted in each smaller section of FIG. 6C.These numbers range from 0 to 90, inclusive. However, these numbers arenot measured numbers; they are merely hypothetical numbers. Moreover,these numbers do not actually exist on the slide 60; these numbersmerely represent the optical densities of the smaller sections 68. Theusable portion of the slide 60 is that portion wherein the values of themeasured optical densities of the smaller sections 68 both (a) exceed alow cut-off value and (b) do not exceed a high cut-off value. As shownin FIG. 6C, the low cut-off value is selected to be zero (0) and thehigh cut-off value is selected to be approximately thirty-eight (38).Therefore, the smaller sections 68 of the usable portion 66 of the slide60 have optical density values ranging from one (1) to thirty-eight(38), inclusive.

The method and device described herein preferably employ scanningdigital microscopy to recognize each of the components in the sample ofwhole blood. A special class of scanning digital microscopy, digitalpathology, is described in greater detail in Digitalpathology-Wikipedia, the free encyclopedia,http://en.wikipedia.org/wiki/Digital_pathology, incorporated herein byreference, and the references and links appended thereto. See alsoScanning Basics 101—All about digital images,http://www.scantips.com/index.html, incorporated herein by reference,and the references and links appended thereto, for additionalinformation about scanning digital images. From the value of the volumeof sample of whole blood deposited on the slide, the method and devicecan determine the parameters described below. Total hemoglobin can bedetermined from the blood smear itself. For example, if two microlitersof blood are dispensed to create the blood smear, and the overallmeasurement of hemoglobin on the slide is 20 g, the absolute volume ofblood in a given area of the blood smear can be determined on the basisof the hemoglobin measured in that given area of the blood smear. Thenumber of white blood cells counted in that same given area of the bloodsmear are counted as cells per unit area and then converted to cells permicroliter.

The concentration of hemoglobin can be calculated from the opticaldensity of the entire scanned blood smear. The determination of opticaldensity can be carried out by means of light having a wavelength of 540nm, which is the maximum absorbance for hemoglobin. However, a differentwavelength (or combination of wavelengths) can be used, if so desired.The same scan for determining optical density can be used for selectingred blood cells and measuring their diameter to provide a measurement ofmean cell diameter, which can be used as a metameter for cell volume.Variability in the mean cell diameter can be used for assessingvariability in the sizes of cells to provide a parameter equivalent tothe red cell distribution width. The absorbance of each red cell withrespect to hemoglobin content permits derivation of a cell by cell andmean hemoglobin content (mean cell hemoglobin) as well as hemoglobinconcentration (mean cell hemoglobin concentration). By measurement ofabsorbance (or optical density), the concentration of hemoglobin of theentire slide can be determined. The quantity of hemoglobin in each ofthe red blood cells (or a statistically significant number of the redblood cells) in the desired area for measurement can be measured. Thismeasurement provides the amount of hemoglobin per red blood cell, orhemoglobin content (CH). A mean value can then be calculated (MCH). Byhaving knowledge of the two-dimensional area of each of the red bloodcells analyzed, the volumes of the individual red blood cells can becalculated. After the volumes of the individual red blood cells areknown, a mean cell volume can be calculated. By using the value of MCH,which is the mean value of the concentration of hemoglobin per red bloodcell, and by using the value of MCV, the value of MCHC, which is theaverage concentration of hemoglobin in a given volume of red bloodcells, can be calculated, i.e., MCH/MCV.

The scan of the red blood cells can be used to determine the presence ofsignificant populations of abnormally shaped cells such as sickle cells,red blood cell fragments, tear drop poikilocytes, acanthocytes,echinocytes, and the like. The scans have the capability of recognizingcellular inclusion bodies, such as, for example, Howell-Jolly bodies,malarial parasites, etc. Atypical aggregates of red blood cells, as seenin Rouleaux formation and cold agglutinin disease, can also be detected.Abnormal patterns of hemoglobin distribution can be detected in caseswhere spherocytes or target cells are present. Fluorochrome stains orsupra-vital staining can be used to detect reticulocytes.

With respect to detection of leukocytes, the method and the devicedescribed herein can employ the staining properties of leukocytes in theentire blood smear to carry out a count of white blood cells. A smallerarea of the blood smear, i.e., that in which the morphologicalcharacteristics of the leukocytes can easily be identified, can be usedto determine the white blood cell differential and to detect and countnucleated red blood cells. The nucleated red blood cells can beidentified on the basis of such features as size, lobularity,granularity (i.e., degree and type of granules), as well as the nuclearand cytoplasmic ratio and morphological characteristics.

Platelets can be counted on the basis of such features as size anduptake of stains. Additionally, interferences in the platelet countcaused by satellitism, and, more commonly, aggregation, can also berecognized. Satellitism means an unusual immune reaction that causesplatelets to stick to neutrophils. When stained and imaged, theplatelets appear to be satellites around the neutrophils. Artifactsresulting from such factors as ageing of the leukocytes in the sample,smear/smudge cells in chronic lymphocytic leukemia, and backgroundstaining seen in cases of paraproteinemia, can also be screened.Smear/smudge cells are ruptured chronic lymphocytic leukemia (CLL) cellsappearing on the blood smears of CLL patients.

The method and the device described herein can be adapted to usefluorochrome detection, thereby providing access to immunofluorescentstaining and uptake of other fluorochrome dyes that can be used fordetection of nucleated cells.

Devices capable of performing morphological scanning and recognition ofcells have been in existence for several years. See, for example,http://www.cellavision.com/?sid=459, incorporated herein by reference.

The following non-limiting examples illustrated specific techniques forcarrying out the method described herein.

EXAMPLE 1

This example illustrates one approach for using a blood smear techniquedescribed herein to carry out a quantitative blood count.

Referring now to FIG. 7, a sample of whole blood 100 drawn from a sampletube “S” and having a known volume is deposited on a microscope slide110 in an initial drop 120, e.g., 50 microliters. It is assumed that noblood is lost in the process for creating the blood smear. Accordingly,all of the cells in the blood sample are accounted for in the bloodsmear. The low power imaging device measures optical density orreflectance resulting from the hemoglobin in a piece-wise fashion (i.e.,via pixels) across the entire area of the blood smear. The low powerimaging device can be a 10X objective lens of a microscope. It isassumed that the optical density or reflectance value of each piece ofthe image (i.e., pixel) is proportional to the amount of red blood cellsin the piece. The term “piece” is synonymous with the smaller section68, shown in FIGS. 6B and 6C. Thus, a piece (or smaller section 68)having an arbitrary response value of ten (10) optical density units orreflectance units would contain twice as many red blood cells as a piece(or smaller section 68) having a response value of five (5) opticaldensity units or reflectance units, so long as the same method is usedto measure each piece (or smaller section 68). The number of measurementunits (e.g., optical density units or reflectance units) recorded overthe entire area of the blood smear is added to yield a numberrepresenting the total quantity of hemoglobin in the blood smear, and,consequently, the total amount of blood in the blood smear is known. Forexample, the total of all non-zero pixels might add up to 10,000,000optical density units or reflectance units.

The next step of the volume calculation is to determine which areas ofthe blood smear are suitable for analysis by means of imaging. Thethicker portions of the blood smear contain too many cells for counting.The thicker portions also contain more hemoglobin and therefore wouldhave higher optical density readings or reflectance readings per pixel.An imaging algorithm for low power imaging can be used to determinewhich areas exhibit an acceptable range for subsequent analysis via highpower imaging. For example, it might be empirically determined thatareas having pixel values in the range of 1 unit through 38 units,inclusive, represent the correct thickness of the blood smear forcounting blood cells, that areas having pixel values of 39 units andgreater represent the portion of the blood smear that is too thick forcounting blood cells, and that areas having pixel values in the range ofless than 1 unit represent the portion of the blood smear that is toothin for counting blood cells. The algorithm would then identify theboundaries of the area of the blood smear where the thickness of theblood smear provides pixel readings in the range of 1 unit through 38units, inclusive. These boundaries would then be used in the high powerimaging step, where only that area within the boundaries is analyzed viahigh power imaging for the purpose of counting cells.

The pixel reference values of all pixels within the bounded measurementarea can be added to obtain a number that is proportional to the totalamount of hemoglobin, and, consequently, the total amount of blood,bounded by the measurement area. For example, the total value of allpixels in the area might add up 3,000,000 optical density units orreflectance units. If the total value of all pixels in the entire smearwere 10,000,000, from the calculation shown previously, then the areabeing analyzed represents the fraction of 3,000,000 divided by10,000,000, which is equivalent to 30% of the total amount of blood, or0.3 times the total amount of blood in the blood smear. Because it isknown that there are 50 microliters of the blood sample in the entireblood smear, the measurement area identified contains 0.3 times 50microliters, or 15 microliters. This volume is then used at a laterpoint in time when the microscopic imaging system records and counts thevarious cells in the identified measurement area. Thus, if that systemdetermines that that are 75,000,000 red blood cells in the areaidentified as being suitable for recording and counting blood cells, thecount of red blood cells for that sample of blood would be 75,000,000per 15 microliters, or 5,000,000 red blood cells per microliter. Thesame calculation can be used to obtain counts of blood cells permicroliter of the blood sample for all other types of cells counted inthe measurement area.

In an alternative method of carrying out the method described in thisexample, if the volume of the whole blood sample applied to the slide isnot known, the volume of the whole blood sample applied can be found bymeans of an independent method of determining the concentration ofhemoglobin. For example, if a total of X grams of hemoglobin is measuredon the slide by means of measuring optical density units or reflectanceunits of the blood smear, and if an independent measurement ofhemoglobin made by an automated hematology analyzer or a spectrometerindicates that the concentration of hemoglobin is X g/dL, simplemathematics would indicate that the volume of the whole blood sampleapplied to the slide is 1 dL. Regardless of how the concentration ofhemoglobin is measured, once the value of the concentration ofhemoglobin is known, the method described herein can be carried out toperform a complete blood count.

EXAMPLE 2

Referring now to FIG. 8A, several different blood smears, ten (10) innumber, are shown. These blood smears represent blood smears made withdifferent samples of whole blood containing varying amounts ofhemoglobin, which causes the overall optical density or reflectance ofthe blood smears to differ in intensity. FIG. 8B shows the thick end ofeach blood smear and the thin end of each blood smear of FIG. 8A. Bymeans of an appropriate scanning procedure, the optical density profileof the blood smear can be graphed. Each point on the X-axis of the graphrepresents the distance of a point of the blood smear on the slide, asmeasured from the point of origin of the drop of whole blood on theslide. The Y-axis of the graph represents the optical density orreflectance at a given point of the blood smear on the slide, asmeasured from the point of origin of the drop of whole blood on theslide. FIG. 8C shows the optical density profiles or reflectanceprofiles of the blood smears in FIG. 8B. FIG. 8D indicates a generaloptical density profile or reflectance profile, wherein the area forcounting blood cells is distinguished from the area where blood cellsare not counted. FIG. 8E illustrates a graph that indicates thecorrelation between hemoglobin measured by means of an automatedhematology analyzer and hemoglobin calculated by scanning a blood smear.

EXAMPLE 3

This example illustrates a technique for determining a blood countwherein only a narrow portion of the minor axis of the microscope slideis used. Instead of using the approach described in Example 1, thevalues corresponding to a line of pixels though the center of a slidecan be measured by the low power imaging system. It should be noted thatthe line being scanned has two dimensions, but the minor dimension ofthe line is much narrower than the minor dimension that is scanned isExample 1. This technique can be used if the device employs a singledetection beam through which the slide is moved, i.e., scanned, whilethe optical density values or reflectance values are recorded. While notbeing a true imaging system, the same result is produced by scanning aline rather than the plurality of lines needed to make up a larger area.It is believed that this embodiment is less expensive that theembodiment described previously.

If the pixel values along the line being scanned, e.g., reflectance oroptical density, are plotted as a function of the position at which themeasurement is made, graphs in the center of the slide are obtained. Theabscissa (X-axis) represents the linear position or distance on the linethrough the middle of the blood smear, and the ordinate (Y-axis)represents the relative optical density or reflectance units measured atthat linear position on the line. The profile on the graph can then beused to identify the point at which the blood smear becomes sufficientlythin to allow recording and counting blood cells by the high powerimaging system. The point on the profile at which the measurements ofreflectance drop below a certain value is the point at which theboundary for the identified measurement area is set. This point isreferred to herein as the cut-off point. That information is thentransmitted to the high power imaging system, which then only recordsand counts cells in the area of the blood smear set by that boundary. Inother words, the “area reviewed” on the slide is the area bounded at theleading edge by a line across the slide, perpendicular to the line bywhich the measured profile was obtained. The perpendicular line acrossthe slide passes through the point identified as the cut-off point. Allpoints on the blood smear that are downstream (i.e., those that are inthe thinner portion of the blood smear) in the blood smear are recordedand counted.

The volume of the area recorded and the position where blood cells arecounted is calculated in a manner that is analogous to that employed inthe embodiment described previously. All the pixel reflectancemeasurements on the scanned line are added, i.e., integrated, to obtaina number representing the total amount of hemoglobin in the blood smear.The quantity of blood in the blood smear can be derived from the totalamount of hemoglobin in the blood smear. The same types of measurementsand calculations as were used in the embodiment described in Example 1are carried out for all points on the profile in the area identified forreview. Again, the proportion of blood from the original sampledesignated by the reviewed areas is determined by recording the value ofthe optical density measured in a particular portion of the blood smear(e.g., a portion of the total area of the blood smear), comparing thatvalue to the optical density of the entire blood smear (i.e., the totalarea of the blood smear), and then determining the blood count on thebasis of the ratio of the value of the optical density measured in theparticular portion of the blood smear (e.g., a portion of the total areaof the blood smear) to the optical density of the entire blood smear(i.e., the total area of the blood smear) while accounting for thevolume of blood forming the blood smear (i.e., a value that is known ora value that can be determined from the total area of the blood smear).The assumption made for this embodiment is that the thickness of theblood smear is uniform along the minor axis of the slide, that is, thedimension perpendicular to the scanned line used by the low powerimaging system to measure the profile. Any asymmetry or uniformity alongthis axis would introduce error in the determination of volume derivedfrom the single axis line through the blood smear.

The method and the device described herein can consolidate the processof blood counting and review of a blood smear in a single instrument.The method and device require only a few reagents, which reagents areinexpensive. The method and device are not complex in a technologicalsense, because only a single undiluted volume of whole blood is used.

Interfering materials, such as, for example, lyse-resistant red bloodcells, would not be a problem. The disposable component is single glassslide. The device is capable of storing its output as an electronicimage. The optics can be arranged to permit fluorescence detection. Thevolume of sample required would be very low.

Because all of the sample would be used for the analysis, the precisionwill be high, particularly when samples of body fluid, moreparticularly, samples of blood, are analyzed. Control materials can belimited to use of reference smears. The method and device describedherein can detect abnormalities that are currently undetectable byconventional hematology analyzers. Such abnormalities include abnormalred blood cell associations (Rouleaux and aggregation), red blood cellinclusion bodies such as Howell-Jolly bodies and malarial parasites. Themethod and device described herein can also show sub-cellular changes inthe white blood cells, such as the Auer rods seen in acute myeloidleukemias or nucleoli seen in blast cells. Finally, the method anddevice described herein can detect plasma abnormalities, such as, forexample, increases in protein levels, which can be seen in cases ofparaproteinemia.

The analysis of the blood cell count and the blood smear can beperformed on the same blood sample, thereby giving the user theopportunity to directly review the instrument's interpretation of theclassification of cells.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

1-22. (canceled)
 23. A device for counting blood cells in a sample ofwhole blood, said device comprising: an aspiration/dispensing componentthat withdraws a sample of whole blood from a container and to depositthe sample of whole blood onto a slide; a spreader that spreads thesample of blood across the slide to create a blood smear; a dryer thatdries the blood smear on the slide; a first imaging system that measuresan absorbance or a reflectance value of light attributable to hemoglobinin a plurality of red blood cells in each of a plurality of sections ofthe blood smear on the slide to determine an optical density for each ofthe sections of the blood smear; a second imaging system that records amagnified, two-dimensional digital image of an area of the blood smear;and a computer comprising a processor and a computer-readable mediumcomprising instructions that, when executed by the processor, cause thedevice to: (a) measure an absorbance or a reflectance value of lightattributable to hemoglobin in a plurality of red blood cells in each ofa plurality of sections of the blood smear on the slide to determine anoptical density for each of the sections of the blood smear; (b)identify an analysis area of the blood smear that has a suitablethickness for analysis; (c) determine an optical density of the entireblood smear; (d) record a magnified two-dimensional digital image of theanalysis area; (e) count a plurality of blood cells in the analysisarea; (f) determine a volume of the sample that is present in theanalysis area by calculating a ratio of the optical density of theanalysis area to the optical density of the entire blood smear andmultiplying the ratio by the volume of the sample; (g) collect, analyzeand store data from the two-dimensional digital image to count aplurality of blood cells in the sample.
 24. The device of claim 23,further including a positioner that positions the slide to enablefurther processing of the blood smear.
 25. The device of claim 23,wherein the device is adapted to deposit a known volume of blood on theslide.
 26. The device of claim 23, wherein the device is adapted tomeasure the concentration of hemoglobin in the sample and the opticaldensity of the blood smear, and to calculate a volume of blood that isdeposited on the slide using the measurements.
 27. The device of claim23, wherein the device is adapted to determine a white blood cell countfrom the blood smear.
 28. The device of claim 23, wherein the device isadapted to determine a platelet count from the blood smear.
 29. Thedevice of claim 23, wherein the device is adapted to determine a redblood cell count from the blood smear.
 30. The device of claim 23,wherein the device is adapted to determine a complete blood count fromthe blood smear.
 31. The device of claim 23, wherein the analysis areaof the blood smear has a thickness that is below an upper cut-off valueand above a lower cut-off value.
 32. The device of claim 31, wherein theupper cut-off value is an optical density of 38, and wherein the lowercut-off value is an optical density of zero.
 33. The device of claim 23,wherein the digital image is a low power digital image.
 34. The deviceof claim 23, wherein the digital image is a high power digital.